Although our current paradigms and treatments for breast and other cancers have resulted in substantial progress, it is apparent the disease frequently evades control and cure. It is therefore imperative that we continue to search for new possibilities in the etiology, progression and treatment of breast cancer. The discovery of stem-like cancer cells has generated tremendous excitement and is offering new paradigms for understanding and treating tumor recurrence and metastasis. The unique physiological environment of solid tumors is characterized by areas of poor flow, hypoxia, high lactate and low pHe that influence a wide-spectrum of characteristics including progression, distant metastasis and response to therapy. Recent studies suggest that hypoxia provides a suitable niche for stem cells to maintain their precursor status. Hypoxia is also a major cause of radiation and chemo-resistance. Our focus in this R21 application is to use molecular and functional imaging to understand the role of hypoxia and physiological and metabolic characteristics in creating or harboring stem-like cancer cells at primary and metastatic sites. The aim and hypothesis will be tested using invasive estrogen receptor/progesterone receptor (ER/PR) negative MDA-MB-231 human and noninvasive ER/PR positive MCF-7 human breast cancer cell lines stably expressing enhanced green fluorescent protein (EGFP) under control of a hypoxia response element (HRE). Co-registered maps of vascular volume, permeability, proton metabolites and extracellular pH will be obtained with MRI. Hypoxic regions will be identified by in vivo optical imaging and fluorescence microscopy. Stem-like cancer cells will be identified in vivo by SPECT imaging of CD44/CD24 expression and immunostaining of fresh/frozen tissue slices. These studies will provide insight into interactions between the tumor microenvironment and cancer stem cells, and may in the future lead to imaging-guided strategies targeting specific microenvironmental niches harboring cancer stem cells. The proposed studies will also identify non-invasive clinically translatable parameters to predict for cancer stem cell harboring regions in tumors. PUBLIC HEALTH RELEVANCE: The discovery of cancer stem cells in several tumors including the breast has generated tremendous excitement and is offering new paradigms for understanding and treating tumor recurrence and metastasis in breast cancer. Here we intend to understand the role of physiological environments such as hypoxia and acidic extracellular pH typically found in tumors in harboring cancer stem cells. The role of hypoxia in acting as a homing signal for cancer stem cells to metastasize to distant locations will also be investigated. These studies will provide new insights into the role of cancer stem cells in recurrence and metastasis.